Estrogen and insulin-like-growth factor 1 (IGF-1) are potent mitogenic stimuli that share important properties in the control of cellular proliferation. However, the coupling between the signaling cascades of estrogen receptors ␣ and ␤ and the IGF-1 receptor (IGF-1R) is poorly understood. Therefore, we selectively transfected estrogen receptor ␣ or ␤ in COS7 and HEK293 cells, which contain IGF-1R. In presence of estrogen receptor ␣ but not ␤, 17␤-estradiol (E2) rapidly induces phosphorylation of the IGF-1R and the extracellular signal-regulated kinases 1/2. Furthermore, upon stimulation with E2, estrogen receptor ␣ but not ␤ bound rapidly to the IGF-1R in COS7 as well as L6 cells, which express all investigated receptors endogenously. Control experiments in the IGF-1R-deficient fibroblast cell line R ؊ showed that after stimulation with E2 only estrogen receptor ␣ bound to the transfected IGF-1R. Overexpression of dominant negative mitogen-activated protein kinases kinase inhibited this effect. Finally, estrogen receptor ␣ but not ␤ is required to induce the activation of the estrogen receptor-responsive reporter ERE-LUC in IGF-1-stimulated cells. Taken together, these data demonstrate that ligand bound estrogen receptor ␣ is required for rapid activation of the IGF-1R signaling cascade.Estrogen as well as insulin-like growth factor 1 (IGF-1) 1 are potent mitogens that are involved in a large array of processes that control proliferation and differentiation in mammalian cells (1, 2). Both mitogens act through receptor-mediated signaling pathways. The cross-talk between these two signaling pathways is currently under investigation (3-6). Estrogen is a steroid hormone that binds to members of the nuclear receptor superfamily (7), whereas IGF-1 as a peptide-growth factor binds to a transmembrane tyrosine kinase receptor, which signals via a series of phosphorylation events (2).Two different estrogen receptors, ER␣ and ER␤, which are encoded by genes located on different chromosomes, have been identified so far (8,9). Sequence analysis demonstrates a high degree of homology between ER␣ and ER␤ in the DNA-binding domain and the ligand-binding domain. However, there are significant differences in regions that would be expected to influence transcriptional activity. The ability of estrogen receptors to activate target gene transcription has been attributed to two regions: the N-terminal activation function 1 (AF-1) and the ligand-dependent AF-2, which is localized in the C-terminal hormone-binding domain (10, 11). AF-1 and AF-2 can activate transcription independently and synergistically, and they act in a promoter-and cell-specific manner (12, 13). Phosphorylation of a serine residue at position 118 is required for full action of the AF-1 (14). Both AF-1 and AF-2 are required to enhance transcription of target genes through AP-1 sites (15). Interestingly, ER␣ and ER␤ act differently at AP-1 sites (16), which may be due to differences in their AF domains (17). ER␣ and ER␤ can form homo-and heterodimers (18), and thus t...

Abstract-Female gender and estrogen-replacement therapy in postmenopausal women are associated with improved heart failure survival, and physiological replacement of 17␤-estradiol (E2) reduces infarct size and cardiomyocyte apoptosis in animal models of myocardial infarction (MI). Here, we characterize the molecular mechanisms of E2 effects on cardiomyocyte survival in vivo and in vitro. Ovariectomized female mice were treated with placebo or physiological E2 replacement, followed by coronary artery ligation (placebo-MI or E2-MI) or sham operation (sham) and hearts were harvested 6, 24, and 72 hours later. After MI, E2 replacement significantly increased activation of the prosurvival kinase, Akt, and decreased cardiomyocyte apoptosis assessed by terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) staining and caspase 3 activation. In vitro, E2 at 1 or 10 nmol/L caused a rapid 2.7-fold increase in Akt phosphorylation and a decrease in apoptosis as measured by TUNEL staining, caspase 3 activation, and DNA laddering in cultured neonatal rat cardiomyocytes. The E2-mediated reduction in apoptosis was reversed by an estrogen receptor (ER) antagonist, ICI 182,780, and by phospho-inositide-3 kinase inhibitors, LY294002 and Wortmannin. Overexpression of a dominant negative-Akt construct also blocked E2-mediated reduction in cardiomyocyte apoptosis. These data show that E2 reduces cardiomyocyte apoptosis in vivo and in vitro by ER-and phospho-inositide-3 kinase-Aktdependent pathways and support the relevance of these pathways in the observed estrogen-mediated reduction in myocardial injury. Key Words: estrogen Ⅲ estrogen receptors Ⅲ myocardial infarction Ⅲ cardiomyocyte Ⅲ apoptosis Ⅲ Akt Ⅲ PI3 kinase H eart failure is a growing public health problem, 1 with several studies demonstrating that women with heart failure have a better prognosis than men. 2,3,4,5,6,7 Whether endogenous sex hormones contribute to these differences in prognosis remains unknown. However, observational studies have demonstrated that postmenopausal women taking estrogen after a myocardial infarction (MI) have a lower incidence of heart failure. 8,9 Furthermore, retrospective analyses of multicenter heart failure trials have shown that postmenopausal women taking estrogen have a better prognosis than women not on estrogen, 10,11 supporting that estrogen may improve heart failure prognosis.Several studies have demonstrated increased cardiomyocyte apoptosis in failing hearts, 12,13 and further evidence suggests that apoptosis contributes to heart failure progression. 14,15 Moreover, autopsy studies have shown that female gender is associated with less cardiomyocyte apoptosis in normal and failing hearts compared with males. 16,17,18,19 These observed gender differences in cardiomyocyte survival provide a plausible explanation for the beneficial effect of female gender on heart failure progression.We recently showed that physiological estrogen replacement in ovariectomized female mice reduces infarct size both early and late after left c...

Taken together, these results show that E2 stimulates the expression of iNOS/eNOS in neonatal and adult cardiomyocytes in-vivo and in-vitro. These novel findings provide a potential mechanism of how estrogen may modulate NOS expression and NO formation in the myocardium.

Physiological effects of estrogen on myocardium are mediated by two intracellular estrogen receptors, ERK K and ERL L, that regulate transcription of target genes through binding to specific DNA target sequences. To define the role of ERL L in the transcriptional activation of both endothelial (eNOS) and inducible nitric oxide synthase (iNOS) in cardiac myocytes, we used the complete ERL L-specific antagonist R,R-tetrahydrochrysene (R,R-THC). R,R-THC inhibited activation of iNOS/eNOS promoter-luciferase reporter constructs (iNOS/eNOS-Luc) in a dose-dependent fashion in COS7 cells selectively transfected with ERL L, but failed to influence ERK K-mediated increase of iNOS/ eNOS-Luc. In neonatal rat cardiomyocytes transfected with eNOS-Luc or iNOS-Luc, incubation with 17L L-estradiol (E2, 10 38 M) for 24 h stimulated expression of eNOS and iNOS. R,R-THC (10 35 M) completely inhibited this effect. Furthermore, eNOS and iNOS protein expression in cardiac myocytes induced by E2 was completely blocked by R,R-THC as shown by immunoblot analysis. Taken together, these results show that ERL L mediates transcriptional activation of eNOS and iNOS by E2. ß